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PUHt INDUSTRY SUB -BRANCH 

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CIRCULAR NO. 454 



Issued January 1938 
Slightly revised May 1945 



UNITED STATES DEPARTMENT OF AGRICULTURE 
WASHINGTON, D. C. 




PREPARATION OF PATHOLOGICAL SPECIMENS FROM 

ANIMAL TISSUES AND THEIR MOUNTING 

UNDER WATCH GLASSES 

By John 8. Bexgston, 1 associate veterinarian, Pathological Division, Bureau of 

Animal Industry 



Introduction 

Selection of tissues and fixation 

Restoring color in specimens 

Removing alcohol and preserving specimens. 



CONTENTS 



Page 
.. 1 

.. 2 
__ 3 

._ 3 



Page 

Mounting specimens under watch glasses 4 

Sealing with asphaltum 7 

Applying the covers 9 

Displaying specimens 12 



INTRODUCTION 

Prior to the last quarter century, glass jars were used almost en- 
tirely in the United States for preserving pathological specimens. 
Although haying the merit of simplicity, this method has several 
rather serious disadvantages. The jars, with the fluid contained in 
them, are heavy, difficult to handle, and easily broken. Specimens so 
preserved cannot be displayed attractively, nor do they photograph 
to advantage. 

In 1913 the mounting of pathological specimens under watch 
glasses, a method which has been practiced in various European 
countries for many years, was introduced into the United States 
through a description by Day. 2 In the preparation of a specimen by 
this method, it is placed under a large convex glass, known as a watch 
glass because of the similarity of its shape to the crystal of a watch 
(fig. 1). A square plate glass (fig. 2) is used as the back. This over- 
comes very largely the disadvantages of the use of glass jars and has 
been used with uniformly good results by members of the veterinary 
and medical profession.' State and city meat inspectors, and veteri- 
nary students. Specimens properly prepared under watch glasses are 
preserved indefinitely, and unless exposed to bright sunlight very 
little fading of color will take place. Specimens prepared more than 
20 years ago are still in perfect condition in the pathological labora- 
tory of the Bureau at Chicago. 

Watch-glass specimens, properly prepared, have proved to be val- 
uable not only for exhibit purposes and other educational use but 



1 In charge of the branch pathological laboratory, Chicago, 111. 
2 Day. L. E. an improved method of mounting museum specimens. 
Vet. Med. Assoc. 44 : 66-71. 1913. 

643953° — 45 



Jour. Amer. 



2 CIRCULAR 454, U. S. DEPARTMENT OF AGRICULTURE 

likewise as authentic scientific evidence of pathological and other ab- 
normal conditions observed from time to time by members of the 
veterinary and medical professions. The purpose of this publication 
is to assist workers in these fields in preparing such specimens in a 
professional manner. 

In mounting specimens under watch glasses it is imperative that 
the beginner follow the detailed technique here described in the 
minutest detail in order to obtain satisfactory results. Each step 
must be painstakingly and properly executed before proceeding to 
the next, otherwise one is likely to encounter difficulties, become dis- 
couraged, and give up the task. The writer cautions all beginners 
against undue haste in the process of mounting specimens under 
watch glasses, but urges slow, careful, and deliberate progress from 
step to step until the technique has been thoroughly mastered. Even 
with the utmost care it will occasionally be necessary to remount 
some specimens a number of times before they can be considered satis- 
factory. One's patience may be sorely tried at times but success will 
ultimately be achieved. 

SELECTION OF TISSUES AND FIXATION 

The tissues or organs selected for mounting under watch glasses 
should be cut or sliced into pieces 1 to iy 2 inches thick and about 6 
inches square, whenever possible. Thin membranous specimens that 
are likely to curl or twist while undergoing fixation should be 
stretched out and tacked to a board of suitable size. All tissues and 
organs selected should be as fresh as possible, as decomposed speci- 
mens are not suitable or satisfactory. The specimens are first rinsed 
in cold water to remove any blood clots or foreign matter that may 
adhere to them, but they should not be allowed to soak in water for 
more than a few minutes. The specimens are then immediately 
placed in a modified Kaiserling fixing solution, known as Kaiserling 
No. 1, which is prepared as follows : Sodium nitrite, 1 g ; potassium 
acetate, 30 g; formalin U. S. P., 200 cc; and distilled water, 1,000 cc. 

This method gives by far the best results for all pathological and 
anatomical material when the color of the hemoglobin in the red 
blood cells is to be preserved. The original Kaiserling No. 1 was 
prepared by using 15 g of potassium nitrate in place of 1 g of sodium 
nitrite, but experience has shown that better color is obtained in the 
specimens by the use of sodium nitrite. Water-white formalin neu- 
tralized with marble chips (calcium carbonate) should be used. 

A liberal quantity of this fluid should be used, and care must be 
taken to place the specimens in the fluid without any twisting, bend- 
ing, or folding, as the tissues will fix in the position in which they 
are placed. It is almost impossible to alter the shape of a specimen 
after fixation, and specimens that are bady twisted or bent cannot 
be mounted satisfactorily. A tight wooden keg provided with a 
cover is the ideal container for this fixing process because wood is 
not attacked by the fixing fluid; all light is excluded, this provision 
being an important one, and the opening of the keg is of sufficient 
size to permit placing the specimens in the fluid in their proper posi- 
tion without bending or twisting. 

The specimens are left in the fixing fluid about 48 hours, when 
they should be removed, washed in cold water a few minutes, and 



PREPARING AND MOUNTING PATHOLOGICAL SPECIMENS 3 

retrimmed to a suitable size, shape, and thickness to fit loosely into 
the watch glass. One should guard against cutting the specimen 
too small, as the finished mount will have a better appearance if the 
specimen practically fills the entire space of the watch glass. The 
thickness of the specimen should be less than the depth of the watch 
glass to be used ; otherwise, when the specimen is immersed in fluid 
between the two glasses air will be trapped therein and will be diffi- 
cult to remove. The specimen should be completely immersed when 
the two glasses are placed together filled with fluid. When the 
tissues have been trimmed to their proper size and thickness, they 
are replaced in the Kaiserling fixing solution for another 24 to 48 
hours to insure thorough fixing. Unless this is done albumin may 
dissolve out in the finished mount, making it cloudy and unsightly. 

RESTORING COLOR IN SPECIMENS 

After the specimens are thoroughly fixed, they are removed from 
the fixing fluid and washed in running water for about 20 minutes 
to remove the surplus fluid. The specimens are then placed in 
60-percent alcohol until the color begins to return. This may take 
from 5 minutes to 1 hour, depending on the tissues. However, they 
must be constantly watched while in the alcohol, and as soon as the 
color begins to return the specimens are removed from the 60-percent 
alcohol and placed in 95-percent alcohol until the color is fully 
restored. This step must be watched closely, because the color will 
begin to fade if the tissues are left in the 95-percent alcohol too long. 
A large glass beaker has been found to be most satisfactory as a 
container for the alcohol and tissues in this step, as the color changes 
can be readily observed. 

REMOVING ALCOHOL AND PRESERVING SPECIMENS 

As soon as the color has been satisfactorily restored to the speci- 
mens, they should be immediately removed from the alcohol and 
placed in a vacuum desiccator of suitable size and quickly covered 
with Kaiserling preservative fluid, known as Kaiserling No. 2, which 
has been previously prepared and stored in a cork-stoppered glass 
bottle. This solution is prepared as follows: Potassium acetate, 
100 g; glycerin C. P. (colorless), 200 cc; and distilled water, 1,000 cc. 

Dissolve the potassium acetate in warm distilled water, add the 
glycerin, bring to a boil in a wide-mouthed pan. filter hot into a 
warm glass bottle, and let cool. 

Cover the specimens ill the desiccator with a perforated porcelain 
desiccator plate, and have sufficient Kaiserling's preservative fluid 
in the desiccator to extend 1 inch above this plate. Expose at once 
to a negative pressure of 20 to 25 inches (50.8 to 63.5 mm) of 
mercury for at least 1 hour, or until the tissues are impregnated 
with the preserving fluid and all the alcohol and air removed. No 
more bubbles should arise from the tissues. Exposing the tissues 
to a negative pressure to remove all alcohol and air is one of the 
most important steps in the entire process of mounting specimens, 
and if omitted or carelessly done the whole process will be a failure. 
It is absolutely essential to remove all traces of alcohol from the 
tissues by negative pressure as soon as they are removed from the 



4 CIRCULAR 454, U. S. DEPARTMENT OF AGRICULTURE 

alcohol. Any delay in doing this will result in fading of color. 
After the alcohol and air have been remoA 7 ed from the tissues they 
may be left in the tightly sealed desiccator until ready to mount 
under watch glasses. 

The materials for mounting these tissue specimens consist of three 
parts: (1) The watch glass, which forms the front of the mount; 
(2) the square plate glass, which forms the back; and (3) book- 
binder's cloth or imitation-leather covered cardboard case, which 
encloses and gives the whole mount its finished appearance. Each 
of these parts requires special consideration. 




Figuke 1. — Colorless flint watch glass. b' inches in diameter, 1 J 4 inches in depth at 
center, and three-sixteenths of an inch thick. 

MOUNTING SPECIMENS UNDER WATCH GLASSES 

Watch glasses may be had in various sizes, but those 6 inches in 
diameter have been found to be most suitable for general use (fig. 1). 
They should be 1 to 1% inches in depth, of even convexity, and of 
colorless flint glass. The thickness of the glass should be not less 
than three-sixteenths of an inch in order that the ground edge of 
the watch glass may have ample contact with the bottom plate 
glass and thus hold firmly and securely when placed in position. 
Watch glasses should be purchased with ground edges but before 
using them it is necessary to grind them perfectly level so that they 



PREPARING AND MOUNTING PATHOLOGICAL SPECIMENS 5 

will fit the plate glass -back in such a manner as to be airtight and 
watertight. This is accomplished by sprinkling fine carborundum 
powder (No. FF) on a large piece of level plate glass (2 by 3 feet 
or larger) and adding a small quantity of water. The grinding is 
performed by moving the watch glass with a rotary motion to and 
fro with moderate pressure until the edge is completely and evenly 
ground. 

The glass used for the back of the specimen must be plate glass. 
The proper size for a 6-inch watch glass is 7% inches square and 
three thirty-seconds of an inch thick (fig. 2). 




Figure 2. — Plate glass for back of mount ; dimensions, 7% inches square and three 
thirty-seconds of an inch thick. 

The ground watch glass and back plate glass are thoroughly 
cleansed by first washing and then immersing in a 20-percent nitric 
acid solution for 15 minutes or longer to remove any greasy film. 
They are then thoroughly rinsed in clean water. The fluid used 
for mounting the specimen between the watch glass and back plate 
glass is Kaiserling No. 2 (preserving fluid) made according to the 
formula already given. Before this fluid is ready for use it should 
be poured into a wide-mouthed pan and quickly brought to a boil 
over a gas flame. The purpose of boiling is twofold: (1) All the 



g CIRCULAR 454, U. S. DEPARTMENT OF AGRICULTURE 

air is driven from the fluid; and (2) it is better to mount the speci- 
men in a warm fluid because as the fluid cools it contracts, drawing 
the watch glass into a closer and tighter contact with the bottom 
plate glass than it would otherwise be possible to obtain. If the 
tissue to be mounted contains fat, care must be taken not to have the 
fluid too warm or the fat will be dissolved and form unsightly 
globules in the finished specimen. 

Remove the specimen from the vacuum desiccator and put it in 
the clean watch glass. Then place the square plate glass on top of the 




Figure 3. — Specimen mounted under watch glass, in Kaiserling No. 2 with all air 

excluded. 

watch glass. Hold the plate glass horizontally in the hands with the 
watch glass, convex side down, containing the specimen, underneath 
the plate glass and slide the watch glass and specimen about one-half 
inch beyond one edge of the plate glass, thus forming a lip. The 
warm Kaiserling Xo. 2 (preserving fluid) is carefully removed from 
the pan with a beaker and poured into the watch glass till it is com- 
pletely full. Then slide the watch glass carefully toward the center 
of the plate glass sufficiently to close the opening. If this has been 
done properly, the watch glass should remain on the plate glass 
where placed without slipping down when held perpendicularly, and 
no fluid should escape. Escape of the fluid indicates that the watch 



PREPARING AND MOUNTING PATHOLOGICAL SPECIMENS 7 

glass has not been ground sufficiently to make a perfect contact with 
the square plate glass. Shake the specimen gently and set aside in a 
perpendicular plane to allow air bubbles to rise to the top. After a 
few minutes slide the watch glass a trifle beyond the edge of the 
plate glass, holding the latter at an angle or almost perpendicularly, 
and pour in sufficient preserving fluid to flush away and replace the 
air bubbles in the watch glass, and again slide the watch glass toward 
the center of the plate glass sufficiently to close the opening. Re- 
peat this operation until no more air bubbles appear within the watch 




Figure 4. — Same as figure 3 after a liberal quantity of melted asphaltum has been 
applied to junction of watch glass and plate glass. 

glass. The watch glass may then be centered on the square plate 
glass and is ready for sealing with melted asphaltum (fig. 3). 



SEALING WITH ASPHALTUM 

It is important to seal the specimen with asphaltum before the 
small quantity of fluid between the ground edge of the watch glass 
and the square plate glass has had time to dry out and be replaced 
by air. Otherwise, the hot asphaltum will drive this air between the 
two glasses into the fluid containing the specimen and produce air 
bubbles. Asphaltum of various degrees of hardness is obtainable, 



8 



CIRCULAR 454, U. S. DEPARTMENT OF AGRICULTURE 



but an asphaltum which neither becomes too brittle in cold weather 
nor too soft in hot weather should be selected. Ordinary asphaltum 
such as is used for street paving has been found to be satisfactory 
for specimen mounting. The asphaltum is melted over a sand bath, 




Figure 5. — Various parts of the cover for enclosing the mounted specimen. A, Inside, 
and B, outside, of front cover, with bookbinder's cloth covering extending 1 inch beyond 
the cardboard (8 inches square, one-sixteenth of an inch thick) on all four sides. The 
circular opening in the center of A and B is 5 l ,4 inches in diameter. C, Middle piece 
of plain cardboard (8 inches square, one-eighth of an inch thick) with a circular 
opening in the center 7 inches in diameter. D, Inside, and E, outside of back cover 
with covering cut flush on all sides of cardboard (8 inches square, one-sixteenth of an 
inch thick). The circular opening in the center of D and E is 5*4 inches in diameter. 

and when it has become thin and fluid it is poured around the joint 
between the two glasses with a teaspoon mounted in a wooden handle. 
A liberal quantity of melted asphaltum should be used; it should 
extend fully one-half inch on the watch glass, and as much on the 



PREPARING AND MOUNTING PATHOLOGICAL SPECIMENS 9 

square plate glass (fig. 4). Inasmuch as melted asphaltum has a 
temperature of 250° F. or more, care must be exercised not to allow 
any of it to come in contact with the skin of the operator or severe 
burns will be produced. 

After the asphaltum has become cool, it can be trimmed as desired 
with a putty knife. The mount is set aside for a day or two to deter- 
mine whether it has been perfectly prepared. Should air bubbles 
appear within the watch glass it should be remounted, which is not a 
difficult task. To remount a specimen remove the greater portion of 




Figure 6. — Front view of completed mount. 



the asphaltum with a putty knife and use a cloth wet with gasoline 
or xylene to remove the last traces. Slide the watch glass up and 
replace the air bubbles with preserving fluid and proceed as before. 



APPLYING THE COVERS 



If the mount has been perfectly prepared it is now ready for 
enclosing in a suitable cover. A firm quality of cardboard about 
one-sixteenth of an inch thick covered on one side with a good grade 
of bookbinder's cloth or imitation leather is very satisfactory for 
enclosing watch-glass specimens to give them a finished and profes- 



10 



CIRCULAR 454, U. S. DEPARTMENT OF AGRICULTURE 



sional appearance. When 7% -inch glass squares and 6-inch Avatch 
glasses are used, the cardboard covers should be 8 inches square. 

A set of covers consists of three parts: (1) A front, (2) back, and 
(3) middle piece (fig. 5). The front and back pieces are made from 
cardboard one-sixteenth of an inch thick and cut into 8-inch squares, 
with a circular opening in the center measuring 5*4 inches in diam- 
eter. Both the front and back pieces of cardboard have the covering 
on one side. For the back of the mount this covering is cut flush 
with the edges of the cardboard used, but on the piece used for the 
front the covering should extend 1 inch beyond the edges of the 
cardboard on all four sides. This allows sufficient covering: to fold 




Figur; 



7. — Portion of wall cases, with cupboard below, used for displaying pathological 
specimens at the branch pathological laboratory, Chicago, 111. 



over the edges of the mount and be glued to the bottom piece. The 
third or middle piece of cardboard is also cut 8 inches square, but it 
should be about one-eighth of an inch thick and have a circular open- 
ing in the center 7 inches in diameter to allow sufficient space for the 
asphaltum used to seal the watch glass to the bottom plate glass. 
No covering is necessary on this piece as it is used inside between the 
plate glass and the front cardboard cover. Covers such as these 
described may be obtained from most good binderies. If it is desired 
to have the name of the specimen stamped in gold letters on the face 
of the specimen cover, it should be done before enclosing the specimen 
therein. Figure 6 shows the front view of a specimen after being 
mounted and enclosed in the cover. 



PREPARING AND MOUNTING PATHOLOGICAL SPECIMENS 



11 



Another method of covering watch-glass specimens has been de- 
vised by Davis 3 of this Bureau. The method is quite different from 
the one just described, both in the materials used and in the prepara- 
tion of covers. The covers, or demountable frames, can be readily 
removed or detached from the specimens. 

The materials used and the technique of the Davis method are 
briefly as follows : The frame, 8 inches square, is made of half-round- 
top picture-frame molding five-sixteenths of an inch wide and 
thirteen-sixteenths of an inch thick with a rabbet seven-sixteenths of 




Fig ore 8. — Portion of wall case containing watch-glass specimens and immediately to the 
right of these, photomicrographs and brief descriptions of the histopathologic^! changes. 



an inch deep and one-eighth of an inch wide. Wood fiberboard 
7% inches square and one-fourth of an inch thick forms the front 
panel with a central opening 5^4 inches in diameter. The panel 
is cut out on its under side so as to accommodate itself to the 
asphaltum applied to the base of the watch glass. Mat board 8 
inches square and one-sixteenth of an inch thick with a central open- 
ing 5*4 inches in diameter is used for the back panel, which is 



8 Davis, C. L. an improved method of framing watch glass museum specimens. 
Arch. Path. 21 : 844. 1936. 



\2 CIRCULAR 454, U. S. DEPARTMENT OF AGRICULTURE 

secured in position by small turn buttons. The frame and panel 
are painted black, and the label is in gold letters stenciled on smooth 
black leather or leatherette affixed to the panel just beneath the 
specimen. 

DISPLAYING SPECIMENS 

To obtain the most benefit from mounted specimens, they should 
be prominently displayed in such a manner as to be readily viewed 
and studied without the necessity of handling them. In the branch 
pathological laboratory at Chicago specimens are displayed in a 
room especially designated for that purpose. This room has a north 
exposure so that direct sunlight never enters it to cause any fading 
of color in the specimens. The mounted watch-glass specimens are 
displayed in special wall cases arranged around the room and in 
large floor cases conveniently placed about the floor of this room. 
The wall cases accommodate four parallel rows of specimens, and 
each specimen is tilted at a 20-degree angle, so that it may be easily 
and comfortably viewed. These cases are placed on the walls at the 
proper height from the floor so as to obviate the necessity of stoop- 
ing or stretching to study the specimens. Each case is provided with 
sliding glass doors to exclude dust. The large floor cases are pro- 
vided with shelves upon which the watch-glass specimens are placed 
in such a manner as to be readily seen from all sides. These cases 
are likewise provided with sliding glass doors. 

Figure 7 shows the method of displaying watch-glass specimens 
in wall cases. These cases are provided with a series of cupboards 
under the specimens where fixing, preserving, and mounting ma- 
terials can be stored. The inside of all wall cases is finished with 
white enamel paint, as a result of which the specimens show up to 
the best advantage. 

Figure 8 shows another wall case that contains, immediately to the 
right of each watch-glass specimen, a photomicrograph and brief 
description of the histopathological changes. These are mounted 
under glass in a narrow black wooden .frame 4 inches wide and 
8 inches high. Specimens displayed in this manner, with a photo- 
micrograph and description of the histopathological changes, are 
exceptionally good for teaching purposes as both the gross and 
microscopic features can be visualized. 



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